Advantages of Chloroplast Transformation Over Nuclear Transformation. The attractiveness of transformation of the chloroplast genome over transformation of the nuclear genome is attributable to the serious risks resulting from the latter. One common concern is the escape of foreign genes through pollen dispersal from transgenic crop plants to their weedy relatives. It has been demonstrated that transgenic pollen will deliver foreign (transgenic) genes to other sexually-compatible plants (detected by marker gene prevalence in progeny harvested from non-transgenic plants grown in surrounding area). For Example, dispersal of pollen from a central test plot containing transgenic cotton plants to surrounding non-transgenic plants has been observed at varying distances in different directions. (Lewellyn and Fitt, 1996); (Umbeck, P. F., et al., 1991). In addition, the frequencies of marker genes in wild sunflowers averaged about 28 to 38%; in wild strawberries growing within 50 meters of a strawberry field, more than 50% of the wild plants contained marker genes from cultivated strawberries. (King, J., 1996).
The escape of foreign genes through pollen is especially a serious environmental concern, in the case of herbicide resistance genes, because of the high rates of gene flow from crops to wild relatives. The concern is that gene escape from transgenic crops to their weedy relatives will create super weeds. In rice (Oryza sativa), gene flow from cultivated varieties to wild relatives has been noted, into O. perennis (Barrett, 1983) and red rice (O. sativa; Langevin et al., 1990). In the southern US, red rice has become a major weed because herbicides that kill it also kills cultivated rice. Decreased prices are paid for cultivated rice contaminated with red rice. Some researchers have introduced the bar gene conferring resistance to glufosinate (Liberty) into cultivated rice to combat this weed (Oard et al., 1996; Sankula et al., 1996). However, due to sexual compatibility, introduction of a nuclear-expressed gene will allow transmission of that resistance trait into red rice via pollen.
Similarly, transgenic oil seed rape, genetically engineered for herbicide resistance outcrossed with a weedy relative, Brassica campestris (field mustard) and conferred herbicide resistance even in the first back-cross generation under field conditions. (Mikkelson, T. R., et al., 1996).
Maternal inheritance of introduced genes prevents gene escape through pollen. Engineering foreign genes through chloroplast genomes (which are maternally inherited for most of the crops) is a solution to this problem. Also, the target enzymes or proteins for most herbicides (e.g. amino acid/fatty acid biosynthetic pathways or photosynthesis) are compartmentalized within the chloroplast. Another important advantage of chloroplast transformation is the higher levels of foreign gene expression due to a very high copy number (5000-10,000) of chloroplast genomes in plant cells. Because the transcriptional and translational machinery of the chloroplast is prokaryotic in nature, herbicide resistant genes of bacterial origin can be expressed at extraordinarily high levels in chloroplasts.
Transformation of the Chloroplast Genome. Early investigations on chloroplast transformation focused on the development of in organello systems using intact chloroplasts capable of efficient and prolonged transcription and translation (Daniell and Rebeiz, 1982; Daniell et al., 1983) and expression of foreign genes in isolated chloroplasts (Daniell and McFadden, 1987). These experiments were done under the premise that it was possible to introduce isolated intact chloroplasts into protoplasts and regenerate transgenic plants (Carlson, 1973). The discovery of the gene gun as a transformation device opened the possibility of direct plastid transformation in plants (Daniell, 1993). Transient expression of foreign genes in plastids of dicots (Daniell et al., 1990; Ye et al., 1990), monocots (Daniell et al., 1991), prolonged foreign gene expression using autonomously replicating chloroplast expression vectors (Daniell et al., 1990) and stable integration of a selectable marker into the tobacco chloroplast genome (Svab and Maliga, 1993) were accomplished using the gene gun. Tobacco plants resistant to certain insects were obtained by integrating the cryIAc gene into the tobacco chloroplast genome (McBride et al., 1995; U.S. Pat. No. 5,451,513, incorporated herein by reference). Stable plastid transformation of higher plants has been accomplished so far only in tobacco.Prior Studies on the Chloroplast Genome. To date, stable integration of a foreign gene into the chloroplast genome of a higher plant has been reported only in tobacco. This was achieved with a vector which was specific for tobacco and which was derived from the tobacco chloroplast genome, that is, the vector contained a sequence homologous only to the tobacco chloroplast genome and which is not highly conserved in the chloroplast genomes of other plants. Such vector is unsuitable for stably transforming plant species other than tobacco. The only published report of foreign gene expression in a plant species other than tobacco is that of wheat leaves and embryos (Daniell et al., 1991), but stable integration was not accomplished. Stable integration of a foreign gene into the chloroplast genome of a monocotyledonous plant has never been reported. At least in cereals (monocots), previously developed transformation/regeneration protocols may not be amenable to plastid transformation due to inherent inefficiencies within those systems. Also, sequential/serial selections (repeated selections), deemed important for achieving homoplasmy (Daniell, 1997), may not be feasible using those regeneration systems employed. Recent development of unique corn (Rudraswamy, 1997) and rice (unpublished) transformation/regeneration protocols have the potential to exhibit substantially increased efficiencies and allow more than one round of selection during regeneration.
Maliga et al. in U.S. Pat. No. 5,451,513 and Svab et al., 1990 propose a transformation of the plastid genome of tobacco by a non-lethal selection technique which employs plastid DNA encoding a non-lethal selectable phenotype. According to Maliga et al. a non-lethal selection is absolutely essential for obtaining transplastgenic lines.
Unlike the Maliga et al. technique, the method of the invention provides a selection which is lethal to all non-transformed plants, but for tobacco. Only the transformed plants survive and continue to grow. This lethal selection takes place with virtually all antibiotics, including spectinomycin and streptomycin in a medium containing the antibiotic in a concentration of 500-1,000 μg/ml. Similar conditions were shown to be non-lethal for tobacco by Maliga et al. Moreover, unlike the technique of Maliga et al., in accordance with the invention, transformation to homoplasmy can be achieved even in the first round of selection.
In European Patent Application No. 0 251 654, Cannon et al. describe transposon-mediated chloroplast transformation of tobacco for instance, using the bacterial transposon Tn5. The vector containing the transposon is targeted at a chromosomal region known to be a “transcriptionally silent” region in order to preserve the transcriptional integrity of the native genes. Such a transcriptionally silent region is identified to be located between two known divergent promoters of chloroplast genes, e.g. the promoters for the genes for the chloroplast large subunit of ribulose bisphosphate carboxylate (RbcL, “LS RuBis Co”) and for β-ATPase (atpB). These promoters transcribe the genes in opposite directions away from the silent region of the chromosome. No transcription terminator is provided in the expression vector of Cannon et al., such terminator regions are known to be absolutely essential for gene expression in plastids. Finally, no stable chloroplast transformation is shown to be accomplished by Cannon et al.
The invention described herein has several distinguishing features over Cannon et al. The invention teaches stable transformation transmittable to the progeny. The integration is not directed into a transcriptionally inactive region of the chloroplast chromosome. The invention integrates a cassette (which contains a transcription terminator as described further hereinafter) into a transcriptionally active region of the chloroplast genome. Promoters controls the expression of one or more genes. Unlike Cannon et al. no transposon is involved in the transformation of the chloroplast in accordance with the invention.
In NATO Asi Series, Daniell et al., 1994 report engineering insect resistance via chloroplast genomes showing the expression of CryII-A protein in plants to control insects. McBride et al, 1995, and U.S. Pat. No. 5,545,818 (1996), confirm report of Daniell et al and show the expression of Bacillus thuringiensis CRYIAc protein into plant plastids. The vectors reported by McBride are designed to introduce the construct only into the tobacco chloroplast genome.
The Need for a Vector to Transform a Variety of Plants. It is evident from the state of the art that an important need exists for a chloroplast integration and expression vector for transforming, preferably stably, the chloroplast genomes of many different species of plants. Such a “universal vector” would permit the transformation of the chloroplast genome of a selected target plant with a heterologous (foreign) DNA coding sequence and eliminate the need to construct vectors which each one is specifically suited to transform the chloroplast genome of the particular plant species which is to be transformed.
The problem to construct such a universal vector competent to transform different plants has to the knowledge of the inventor, not yet been solved.
Prior Art Concepts of the Intergenic Spacer Region. While the nucleotide sequence of coding regions of the genome, including the chloroplast genome, are often conserved between species, in contrast the sequences flanking functional genes, i.e. the spacer regions between coding regions typically are not conserved. The accepted dogma for lack of conservation, and thus the low degree of homology between species of spacer regions, is that the spacer regions typically do not perform essential functions. Therefore, there is little, if any, selective pressure to conserve the sequence of spacer regions between species. The sequence of the spacer regions may be altered without undesirable effects.
Stummann et al., 1988, disclose that the gene order of the ribosomal RNA operon of the chloroplast genome is the same between different species of plants, including tobacco, maize, and a liverwort, Marchantia, and that the coding sequences of this operon are highly homologous. Stummann also discloses that the interspecies homology of the operon is less than the interspecies homology of the gene coding regions. This is consistent with the lack of conservation of spacer regions; and suggests that the interspecies homology of spacer regions in the ribosomal RNA operon is relatively low.
The invention, contrary to the dogma of lack of conservation of the spacer regions, uses spacer regions that are highly conserved between different plants to construct vectors competent to transform a variety of plants.